In the United States cardiovascular disease is the major cause of disabilities and accounts for approximately half of all deaths each year. The disease is complex with both genetic and epigenetic factors influencing its severity. Moreover, the genetic factors are not well understood due to the number of genes involved and the difficulty in accounting for the multiple environmental influences effecting the rate of cardiovascular disease on the normal population. Because of its complexity it would be of great benefit to have animal systems, were the environmental effects can be carefully controlled, with known modification in genes thought to be involved in the development of atherogenesis. Recently the ability to modify specific genes in mouse embryonic stem cells by homologous recombination, combined with the ability of the modified ES cells to contribute to the formation of the germ cells in ES- Blastocyst chimeras, has opened a new venue with which to generate animal models of cardiovascular disease. This technology has been utilized by us to generate a mouse model of familial typeIII hyperlipo-proteinemia and premature atherosclerosis by inactivation of the apolipoprotein E gene. It would be of great interest to generate a porcine model with apoE deficiency to complement the existing mouse model. Unavailability of stable ES cell lines from forcing origin prevents us from doing so, however. The overall objective of this application include: 1) the isolation and characterization of stable cell line of porcine embryonic stem cells. Porcine ES cells will be isolated and maintained in an undifferentiated state by addition of recombinant porcine leukemia inhibitory factor (pLIF) to the culture media. Isolated cell lines will be characterized for their ability to differentiate in vitro and for their ability to generate germ line chimeras; 2) the cloning and molecular characterization of the porcine apolipoprotein-E gene. The apoE gene will be isolated from porcine genomic library and sequenced to determined overall homology to the human and the mouse gene. Additionally, DNA polymorphisms at the apoE locus will be determined by single stranded conformational polymorphisms and heteroduplex DNA analysis; 3) development of a porcine model of familial type III hyperlipoproteinemia. The inactivation of the porcine apoE gene will be accomplished by a homologous recombination event resulting in insertion of the neo gene in exon 2. The resulting animal model will not only complement the existing mouse model of apoE deficiency but will also provide the opportunity for looking at phenotypic changes due to apoE deficiency that are either not evident in mice or difficult to study in a small animal model such as the mouse.